The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

Abstract

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, we decided to investigate the possible relationship between these two phenomena. Steroid-free GR was converted from a 9S to a 4S form by exposure to 0.4 M NaCl. The binding of [3H]triamcinolone acetonide [( 3H]TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas [3H]TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR exposed to 0.4 M NaCl in the presence of 10 mM molybdate showed the presence of two types of binding sites with apparent dissociation constants of 0.52 +/- 0.07 and 64.1 +/- 16.2 nM, respectively. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low [3H]TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. Immunoblot analysis using anti-GR monoclonal antibody revealed no difference in molecular size (Mr 94000) between the high- and low-affinity entities. These results indicate that the transformed GR has a reduced [3H]TA-binding affinity as compared to the nontransformed GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct Roles of the Molecular Chaperone hsp90 in Modulating Dioxin Receptor Function via the Basic Helix-Loop-Helix and PAS Domains

“…Specific ligand-binding activity of in vitrotranslated proteins was determined as previously described (25), by using [ 3 H]dioxin (40 Ci/mmol; Chemsyn) as a ligand, with the exception that the washing steps were carried out in the presence of 0.5% Tween 20. The DNA-binding activity of the dioxin receptor was monitored by a gel mobility shift assay performed essentially as described previously (16,28). Briefly, DNA-binding reaction mixtures were assembled with the indicated in vitro-translated proteins or cellular extracts in 10 mM HEPES, (N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid) (pH 7.9)-5% (vol/vol) glycerol-0.5 mM dithiothreitol-2.5 mM MgCl 2 -1 mM ethylene diaminetetraacetic acid-0.08 (wt/vol) Ficoll at a final concentration of 50 mM NaCl and in a final volume ranging between 30 and 50 l. A 36-bp 32 P-3Ј-end-labeled, double-stranded oligonucleotide XRE (5), spanning the dioxin-responsive XRE1 element of the rat cytochrome P-4501A1 promoter region (13), was added to the reaction mixtures as a specific probe in the presence of 1 g of poly(dI-dC) (Pharmacia) nonspecific competitor DNA.…”

The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo.

“…For some cases, functional significance of the complex formation of HSP90 and its target protein has been demonstrated. The affinity of glucocorticoid receptors to the ligands is a hundred times higher in the complex with HSP90 than in its HSP90-free form (20). Furthermore, activation of glucocorticoid receptors by the ligands in yeast cells requires the expression of a sufficient amount of HSP90 (24).…”

Interaction between the N-terminal and Middle Regions Is Essential for the in Vivo Function of HSP90 Molecular Chaperone

“…At higher concentrations, 8-and 19-kDa fragments accumulated. Among them, the 14-kDa fragment was the most resistant to a wide range of trypsin concentrations (lanes [2][3][4][5][6].…”

“…The 90-kDa heat shock protein (HSP90) 1 has been demonstrated to be an important molecule, chaperoning a variety of cellular proteins, such as steroid receptors (1)(2)(3), protein kinases involved in signal transduction (4 -6), and even retrovirus reverse transcriptase (7) and endothelial nitric-oxide synthase (8) (for reviews, see Refs. 9 and 10).…”